Fathima Sheriff

17-Beta-estradiol induces transformation and tumorigenesis in human breast epithelial cells

Breast cancer is a malignancy whose dependence on estrogen exposure has long been recognized even though the mechanisms whereby estrogens cause cancer are not clearly understood. This work was performed to determine whether 17β‐estradiol (E2), the predominant circulating ovarian steroid, is carcinogenic in human breast epithelial cells and whether nonreceptor mechanisms are involved in the initiation of breast cancer. For this purpose, the effect of four 24 h alternate periods of 70 nM E2 treatment of the estrogen receptor alpha (ER‐α) negative MCF‐10F cell line on the in vitro expression of neoplastic transformation was evaluated. E2 treatment induced the expression of anchorage‐independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, and loss of 9p11–13. Only invasive cells that exhibited a 4p15.3–16 deletion were tumorigenic. Tumors were poorly differentiated ER‐α and progesterone receptor‐negative adenocarcinomas that expressed keratins, EMA, and E‐cadherin. Tumors and tumor‐derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3–13, 8p11.1–21, 9p21‐qter, and 18q, and gains in 1p, and 5q15‐qter. The induction of complete transformation of MCF‐10F cells in vitro confirms the carcinogenicity of E2, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.—Russo, J., Fernandez, S. V., Russo, P. A., Fernbaugh, R., Sheriff, F. S., Lareef, H. M., Garber, J., Russo, I. H. 17‐Beta‐estradiol induces transformation and tumorigenesis in human breast epithelial cells. FASEB J. 20, 1622–1634 (2006)

Molecular basis of pregnancy-induced breast cancer protection.

We have postulated that the lifetime protective effect of an early pregnancy against breast cancer is due to the complete differentiation of the mammary gland characterized by a specific genomic signature imprinted by the physiological process of pregnancy. In the present work, we show evidence that the breast tissue of postmenopausal parous women has had a shifting of stem cell 1 to stem cell 2 with a genomic signature different from similar structures derived from postmenopausal nulliparous women that have stem cell 1. Those genes that are significantly different are grouped in major categories on the basis of their putative functional significance. Among them are those gene transcripts related to immune surveillance, DNA repair, transcription, chromatin structure/activators/co-activators, growth factor and signal transduction pathway, transport and cell trafficking, cell proliferation, differentiation, cell adhesion, protein synthesis and cell metabolism. From these data, it was concluded that during pregnancy there are significant genomic changes that reflect profound alterations in the basic physiology of the mammary gland that explain the protective effect against carcinogenesis. The implication of this knowledge is that when the genomic signature of protection or refractoriness to carcinogenesis is acquired by the shifting of stem cell 1 to stem cell 2, the hormonal milieu induced by pregnancy or pregnancy-like conditions is no longer required. This is a novel concept that challenges the current knowledge that a chemopreventive agent needs to be given for a long period to suppress a metabolic pathway or abrogate the function of an organ.

Characterization of a Genomic Signature of Pregnancy Identified in the Breast

The objective of this study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy. The study was designed as a two-phase approach. In the discovery phase, we compared breast genomic profiles of 37 parous with 18 nulliparous postmenopausal women. In the validation phase, confirmation of the genomic patterns observed in the discovery phase was sought in an independent set of 30 parous and 22 nulliparous postmenopausal women. RNA was hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays containing probes to 54,675 transcripts, scanned and the images analyzed using Affymetrix GCOS software. Surrogate variable analysis, logistic regression, and significance analysis of microarrays were used to identify statistically significant differences in expression of genes. The false discovery rate (FDR) approach was used to control for multiple comparisons. We found that 208 genes (305 probe sets) were differentially expressed between parous and nulliparous women in both discovery and validation phases of the study at an FDR of 10% and with at least a 1.25-fold change. These genes are involved in regulation of transcription, centrosome organization, RNA splicing, cell-cycle control, adhesion, and differentiation. The results provide initial evidence that full-term pregnancy induces long-term genomic changes in the breast. The genomic signature of pregnancy could be used as an intermediate marker to assess potential chemopreventive interventions with hormones mimicking the effects of pregnancy for prevention of breast cancer. Cancer Prev Res; 4(9); 1457–64. ©2011 AACR.

Histologic changes in the breast with menopausal hormone therapy use: correlation with breast density, estrogen receptor, progesterone receptor, and proliferation indices.

Objective: This retrospective study systematically compared mammographic density with histology in women receiving or not receiving menopausal hormone therapy (HT). Design: This study was approved by the institutional review board. Twenty-eight postmenopausal women using HT were matched with 28 postmenopausal women not using HT at the time of breast cancer diagnosis. Noncancerous tissue from mastectomy specimens was examined histologically to quantitate the content of fibrous stroma, ducts, and lobule types 1, 2, and 3. Tissue samples were also evaluated for estrogen receptor, progesterone receptor, and Ki67 activity in the ducts and lobules. Breast density was quantified by digitizing the contralateral mammogram and computer-assisted interactive thresholding. Results: High breast density in women using HT was correlated with greater fibrous stroma (P = 0.020) and lobule type 1 (P = 0.016). Breast density also correlated with Ki67 activity in the ducts (P = 0.031) and lobules (P= 0.023) for both groups combined. Estrogen and progesterone receptors did not correlate with either breast density or HT use. Conclusions: Increased fibrous stroma and lobule type 1 are associated with increasing mammographic density in women using HT, independent of estrogen and progesterone receptor up-regulation. These findings suggest that increased breast density may be mediated through a paracrine effect. The increase in breast cancer risk with HT use may be due to an increase in target lobule type 1 cells.

Formation of depurinating N3Adenine and N7Guanine adducts by MCF-10F cells cultured in the presence of 4-hydroxyestradiol

Metabolic conversion of endogenous estrogens, estradiol (E2) and estrone (E1), to the catechol estrogens 4‐hydroxyE1(E2) [4‐OHE1(E2)] has been implicated in the initiation of cancer in rodents and humans. Evidence collected in our laboratories has shown that 4‐OHE1(E2) are enzymatically oxidized to E1(E2)‐3,4‐quinones [E1(E2)‐3,4‐Q], which have the potential to damage DNA by forming predominantly depurinating adducts, 4‐OHE1(E2)‐1‐N3Ade and 4‐OHE1(E2)‐1‐N7Gua, leading to the accumulation of mutations and probably cell transformation. The human breast epithelial cell line MCF‐10F has been transformed by treatment with E2 or 4‐OHE2. We have used MCF‐10F cells to study the presence of adducts and conjugates after treatment with 4‐OHE2. To mimic the intermittent exposure of breast cells to endogenous estrogens, MCF‐10F cells were treated with 1 μM 4‐OHE2 for a 24‐h period at 72, 120, 192 and 240 h postplating. Culture media were collected at each point, extracted by solid‐phase extraction and analyzed by HPLC connected with a multichannel electrochemical detector and/or ultraperformance liquid chromatography/tandem mass spectrometry. Media from successive treatments with 4‐OHE2 showed the formation of methoxy and cysteine conjugates, and the depurinating adducts 4‐OHE1(E2)‐1‐N3Ade. The amount of 4‐OHE1(E2)‐1‐N3Ade adducts was higher after the third treatment; smaller amounts of the 4‐OHE1(E2)‐1‐N7Gua adducts were detected after the second and third treatments. These results demonstrate that MCF‐10F cells oxidize 4‐OHE2 to E1(E2)‐3,4‐Q, which react with DNA to form the depurinating N3Ade and N7Gua adducts. This DNA damage can play an important role in the 4‐OHE2‐induced mutations and transformation of MCF‐10F cells to malignant cells.

Pregnancy-induced chromatin remodeling in the breast of postmenopausal women.

Early pregnancy and multiparity are known to reduce the risk of women to develop breast cancer at menopause. Based on the knowledge that the differentiation of the breast induced by the hormones of pregnancy plays a major role in this protection, this work was performed with the purpose of identifying what differentiation‐associated molecular changes persist in the breast until menopause. Core needle biopsies (CNB) obtained from the breast of 42 nulliparous (NP) and 71 parous (P) postmenopausal women were analyzed in morphology, immunocytochemistry and gene expression. Whereas in the NP breast, nuclei of epithelial cells were large and euchromatic, in the P breast they were small and hyperchromatic, showing strong methylation of histone 3 at lysine 9 and 27. Transcriptomic analysis performed using Affymetrix HG_U133 oligonucleotide arrays revealed that in CNB of the P breast, there were 267 upregulated probesets that comprised genes controlling chromatin organization, transcription regulation, splicing machinery, mRNA processing and noncoding elements including XIST. We concluded that the differentiation process induced by pregnancy is centered in chromatin remodeling and in the mRNA processing reactome, both of which emerge as important regulatory pathways. These are indicative of a safeguard step that maintains the fidelity of the transcription process, becoming the ultimate mechanism mediating the protection of the breast conferred by full‐term pregnancy.

Defining the genomic signature of the parous breast

BackgroundIt is accepted that a womans lifetime risk of developing breast cancer after menopause is reduced by early full term pregnancy and multiparity. This phenomenon is thought to be associated with the development and differentiation of the breast during pregnancy.MethodsIn order to understand the underlying molecular mechanisms of pregnancy induced breast cancer protection, we profiled and compared the transcriptomes of normal breast tissue biopsies from 71 parous (P) and 42 nulliparous (NP) healthy postmenopausal women using Affymetrix Human Genome U133 Plus 2.0 arrays. To validate the results, we performed real time PCR and immunohistochemistry.ResultsWe identified 305 differentially expressed probesets (208 distinct genes). Of these, 267 probesets were up- and 38 down-regulated in parous breast samples; bioinformatics analysis using gene ontology enrichment revealed that up-regulated genes in the parous breast represented biological processes involving differentiation and development, anchoring of epithelial cells to the basement membrane, hemidesmosome and cell-substrate junction assembly, mRNA and RNA metabolic processes and RNA splicing machinery. The down-regulated genes represented biological processes that comprised cell proliferation, regulation of IGF-like growth factor receptor signaling, somatic stem cell maintenance, muscle cell differentiation and apoptosis.ConclusionsThis study suggests that the differentiation of the breast imprints a genomic signature that is centered in the mRNA processing reactome. These findings indicate that pregnancy may induce a safeguard mechanism at post-transcriptional level that maintains the fidelity of the transcriptional process.

Gene expression profile induced by pregnancy in the breast of premenopausal women

We previously reported that having completed a full term pregnancy (FTP) confers specific gene expression patterns in the breast of healthy postmenopausal women [Belitskaya-Levy, I. et al. 2011, Peri, S. et al. 2012 and Russo, J. 2012]. In the present work, we report on gene expression differences in the breast of parous versus nulliparous healthy premenopausal women. Using Affymetrix Human Genome U133 Plus 2.0 microarrays, we analyzed the gene expression profile of breast tissue from 30 nulliparous (NP) and 79 parous (P) premenopausal volunteers between the ages of 30 and 47 years who were free of breast pathology. Because of the known short-term increase in breast cancer risk preceding the long-term protective effect of FTP, we also examined gene expression differences in P vs. NP women as a function of time since last FTP. Through multiple regression analysis, controlling for confounders, we found 416 probesets differentially expressed (fold-change ≥ 1.2 and false discovery rate Citation Format: Julia Santucci-Pereira, Anne Zeleniuch-Jacquotte, Yelena Afanasyeva, Hua Zhong, Eric A. Ross, Michael Slifker, Suraj Peri, Ricardo Lopez de Cicco, Yubo Zhai, Irma H. Russo, Theresa Nguyen, Fathima Sheriff, Alan A. Arslan, Pal Bordas, Per Lenner, Janet Ahman, Anna-Stina L. Eriksson, Robert Johansson, Goran Hallmans, Paolo Toniolo, Jose Russo. Gene expression profile induced by pregnancy in the breast of premenopausal women. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2360. doi:10.1158/1538-7445.AM2014-2360

Methodological Approach to Tissue Microarray for Studying the Normal and Cancerous Human Breast

Tissue microarray (TMA) technology is a tool that allows for the rapid evaluation of a large number of tissue samples on a single section of a microscope slide [1]. Cores of paraffin embedded tissue are removed from the original donor block and re-embedded into a recipient block in an ordered pattern of hundreds of cores. The cores can be from a variety of normal and abnormal types of tissue or stages of tumor and can be used to survey marker expression. This is more expedient than staining and scoring full sections from each donor block; it conserves the reagents used in the assay (e.g., immunohistochemistry or fluorescent in situ hybridization) [1–9], and extends the use of the original donor blocks. Many techniques for preparing the TMAs are in use with variable performance characteristics depending upon the tissue type, the coring procedure, and instrumentation (Fig. 4.1). The advantage of TMA is that it allows for examination of markers with clinical features, associations to be made between different markers and of markers with response to therapy or prognosis, and assessment of markers for the purpose of guiding treatment.

Methodology for Studying the Compartments of the Human Breast

A shortcoming of using human breast tissue for molecular analysis is the heterogeneous nature of the sample. To resolve this, various microdissection techniques have been employed to obtain homogeneous cells, such as manual scraping of tissue with scalpel blades, needles, or other probes to positively select cells of interest. These techniques are limited because of poor delineation of tissue and high susceptibility to contamination from dissimilar cells. Infrared laser capture microdissection (LCM), developed at the National Institute סf Health, has become increasingly commercially available in the last two decades of the twentieth century [1–3]. Laser capture microdissection, also called microdissection, laser microdissection (LMD), is a method for isolating cells or specific regions of interest from cells, tissue, or organisms. This technique enables researchers to investigate DNA/RNA and proteins from specific cells or regions of tissue. LCM consists of an LMD system, camera, and software used to select and collect the areas of interest. The basic principle of LCM involves a laser which fuses the desired material onto a specialized cap which can be then close over a 0.5 mL microcentrifuge tube. Before LCM can be performed, a paraffin-embedded tissue (PET) is sectioned with a regular microtome, or a cryostat is used to create histological sections from frozen tissue. The histological sections are placed onto a membrane slide which is then freshly hemotoxylin and eosin (H&E) stained. After the slide has been stained, the sample is set in 100 % EtOH followed by air drying.